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Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof

A technology of fusion protein and Escherichia coli, which is applied in the direction of chemical instruments and methods, and the use of vectors to introduce foreign genetic material, bacteria, etc., can solve the problems of cumbersome purification methods of adhesin, easy cross-reaction, low purity and yield, and achieve immunity Good reactivity, low cost, and high yield

Active Publication Date: 2015-11-18
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, bacteria were often used as the coating antigen. Although the cost is low, the purity and yield are low, and the specificity is poor, and cross-reaction is easy to occur. Some researchers use purified adhesive pili as the coating antigen, which has good specificity. However, the purification method of adhesin is cumbersome

Method used

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  • Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof
  • Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof
  • Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of fusion protein for detection of anti-ETEC antibodies

[0023] 1. Fusion gene designed to detect anti-ETEC antibody

[0024] The nucleotide and amino acid sequences of ETECF4(K88) pili and ETECF5(K99) pili were analyzed by the biological software DNAStar. After analysis using the codon optimization software CodonAdaptationTool (JCAT), replace the rare codons of E. coli in the nucleotide sequences of F4 and F5 pili with preferred codons, and add a linker sequence between the gene fragments of F4 and F5 pili, The fusion gene was designed, and the sequence is shown in SEQ ID NO:2. Add an EcoRI restriction site at the 5' end of the fusion gene, add a SalI restriction site at the 3' end, and send it to Nanjing GenScript Biotechnology Company for synthesis.

[0025] The fusion genes encode fusion proteins F4-F5 for detection of anti-ETEC antibodies. The amino acid sequence of the fusion protein F4-F5 is shown in SEQ ID NO:1.

[0026] 2. Constructio...

Embodiment 2

[0039] Embodiment 2 Detects the composition and method of use of the anti-ETEC antibody kit

[0040] 1. The composition of the kit

[0041] 1. Washing solution: prepare pH 7.4, 0.01M phosphate buffer solution, and add Tween-20 with a final concentration of 0.05%. Specific preparation method: weigh KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 Dissolve O2.9g and NaCl8g in water, then dilute to 1000mL, add 0.5mL Tween-20.

[0042] 2. Diluent: pH7.4, 0.01M phosphate buffer, the preparation method is the same as above.

[0043] 3. Stop solution: 2M sulfuric acid solution, dilute 111.2mL of concentrated sulfuric acid with water and dilute to 1000mL.

[0044] 4. Substrate chromogenic solution: soluble TMB one-component substrate chromogenic solution (purchased from Nanjing Zhuyan Biotechnology Co., Ltd.).

[0045] TMB is an abbreviation for 3,3’,5,5’-tetramethylbenzidine.

[0046] 5. ETEC-positive serum standard product: from multiple ETEC-immunized piglet sera, select a micro-agglutina...

Embodiment 3

[0080] Embodiment 3 specificity experiment

[0081] Investigate the specificity of the kit in Example 2. Adopt kit in embodiment 2 to know PEDV (porcine epidemic diarrhea virus), PRRSV (porcine reproductive and respiratory syndrome virus), PRV (pseudorabies virus), CSFV (swine fever virus), PCV2 with indirect ELISA method (Porcine circovirus type 2), APP (Actinobacillus pleuropneumoniae) and SS (Streptococcus suis) positive sera were tested. Three parallel replicates were performed for each serum. The OD value of each well was measured with a microplate reader at a wavelength of 450 nm to determine the specificity of the indirect ELISA method. The positive sera of PEDV, PRRSV, PRV, CSFV, and PCV2 were purchased from IDEXX Company, and the positive sera of APP and SS were purchased from Wuhan Keqian Biotechnology Co., Ltd. The results showed that the S / P of PEDV, PRRSV, PRV, CSFV, FMDV, PCV2, APP, and SS positive sera were all less than 0.23, which was judged as negative.

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Abstract

The invention provides a fusion protein for detecting an anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as a preparation method and application thereof, and belongs to the technical field of biology. The fusion protein has an amino acid sequence as shown in SEQ ID NO:1. The method for preparing the fusion protein comprises the following steps: inserting a coding gene of the fusion protein into a pCold I plasmid to obtain a recombinant vector; transforming a host cell by the recombinant vector to obtain a positive recombinant bacterium; and inducing the positive recombinant bacterium to express the fusion protein, and purifying to obtain the fusion protein. The fusion protein has excellent immunoreactivity and high yield, can be used for simultaneously detecting the anti-F4 type ETEC and anti-F5 type ETEC antibody of pigs. A kit for detecting the anti-ETEC antibody is exact in detection, simple and convenient to operate and excellent in specificity, sensibility and repeatability, is applicable to popularization in clinical application, and provides a reliable means to rapid detection of the anti-ETEC antibody.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion protein, a preparation method and an application for detecting antibodies against porcine enterotoxigenic Escherichia coli. Background technique [0002] Enterotoxigenic Escherichia Coli (ETEC) is the most common pathogenic Escherichia coli with diarrhea in humans and young animals (newborn piglets, calves, lambs and weaned piglets), and its pathogenicity is mainly composed of adhesin pili and enterotoxin It consists of two virulence factors, which are closely related and indispensable. ETEC first adsorbs to receptors on the surface of small intestinal mucosal epithelial cells through specific adhesin, and colonizes on the intestinal surface. Newborn young animals died of severe watery diarrhea and rapid dehydration after being infected by ETEC, with high morbidity and mortality. According to statistics, domestic young animal ETEC diarrhea accounts for 35% of p...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/52C12N15/70C12N1/21G01N33/68G01N33/569
Inventor 张雪寒俞正玉张强张碧成汪伟茅爱华周俊明何孔旺倪艳秀温立斌李彬周萍
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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